The native ORAI channel trio underlies the diversity of Ca2+ signaling events

Abstract

The essential role of ORAI1 channels in receptor-evoked Ca 2+ signaling is well understood, yet little is known about the physiological activation of the ORAI channel trio natively expressed in all cells. The roles of ORAI2 and ORAI3 have remained obscure. We show that ORAI2 and ORAI3 channels play a critical role in mediating the regenerative Ca 2+ oscillations induced by physiological receptor activation, yet ORAI1 is dispensable in generation of oscillations. We reveal that ORAI2 and ORAI3 channels multimerize with ORAI1 to expand the range of sensitivity of receptor-activated Ca 2+ signals, reflecting their enhanced basal STIM1-binding and heightened Ca 2+-dependent inactivation. This broadened bandwidth of Ca 2+ influx is translated by cells into differential activation of NFAT1 and NFAT4 isoforms. Our results uncover a long-sought role for ORAI2 and ORAI3, revealing an intricate control mechanism whereby heteromerization of ORAI channels mediates graded Ca 2+ signals that extend the agonist-sensitivity to fine-tune transcriptional control.

Omnitemporal choreographies of IP3R and all five STIM/Orai underlie the complexity of mammalian Ca2+signaling

“…3A, B ). In agreement with previous data from our lab 28 , we showed that under conditions of stimulation with low concentrations of CCh (10 µM), relieving the negative regulatory effects of Orai2 and Orai3 on native Orai1 results in enhanced percentage (∼70%) of Orai2,3-KO cells responding with Ca 2+ plateaus ( Fig. 3C, Q-S; compare to 16% of WT-HEK293 cells in Fig.…”

“…Furthermore, Orai-TKO cells have a significantly reduced frequency of oscillations with only ∼3 oscillations/14 min ( Fig. 1H-I see also 28 ). These findings suggest that STIM proteins provide an additional control mechanism over the rate of ER Ca 2+ release through IP 3 Rs.…”

“…Orai1/2/3 triple knockout cells (Orai-TKO), which show complete abrogation of SOCE 28 , were included as controls. In our previous studies, we determined that 10 μM of the muscarinic receptor agonist carbachol (CCh) induces a sustained Ca 2+ oscillatory response 28 . For each condition, five representative traces from individual cells are shown ( Fig.…”

“…We recently provided evidence supporting the notion that native CRAC channels are heteromers of Orai isoforms. Without Orai2 and Orai3, physiological Ca 2+ oscillations typically obtained in response to low- and mid-range agonist concentrations are mostly replaced with Orai1-driven Ca 2+ plateaus 28 . Here, we performed side by side Förster resonance energy transfer (FRET) experiments with either STIM1-YFP or STIM2-YFP co-expressed with each CFP-Orai isoform in Orai-TKO cells ( Fig.…”

“…In most cells however, Ca 2+ oscillations are initiated by IP 3 R-mediated Ca 2+ release and maintained by ER Ca 2+ store refilling through SOCE 26,27 . We recently demonstrated that Orai1 is dispensable for sustaining regenerative Ca 2+ oscillations in HEK293 cells 28 . Surprisingly, endogenous Orai2 and Orai3, which contribute almost undetectable fraction of SOCE were able to maintain oscillations, suggesting that miniscule levels of SOCE are enough to support Ca 2+ oscillations over extended periods of time 28 .…”